The present invention relates to the production of biomass for the isolation of ccc plasmid DNA comprising culturing a bacterial transformant in a bioreactor containing an antibiotic-free batch medium under batch-conditions and, at the end of the batch phase, feeding under feed-back conditions a portion of a feed medium after the rise of DO above a threshold-set point. Said feed medium comprises besides a carbon and a nitrogen source, a magnesium salt, preferably in concentrations above 20 mM. Preferably, the bacterial transformant is harvested after the end of the culture and frozen or freeze-dried. Also preferred is that ccc plasmid DNA is, optionally directly, isolated after harvesting the bacteria.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer""s specifications, instructions, etc.) are hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the present invention.
With the advent and progress of recombinant DNA technology into a variety of fields such as food stuff production and medical therapy, the desire for large quantities of highly pure DNA has constantly risen. Traditional methods of purifying genomic or plasmid DNA (see, e.g., Sambrook et al., xe2x80x9cMolecular Cloning, A Laboratory Manualxe2x80x9d, CSH Press, 2nd edition, 1989, Cold Spring Harbor N.Y.) usually require sophisticated methodology if the DNA is to be free from RNA and other contaminating organic compounds. In particular, methods for obtaining ccc plasmid DNA in pure form regularly suffer from the disadvantage that other plasmid topologies also produced have to be separated from the desired product. For example, Lahijani et al., Human Gene Therapy 7 (1996), 1971-1980 have reported that high yields of pBR322-derived plasmids intended for human gene therapy may be obtained when plasmids comprising a temperature-sensitive single-point mutation that affects the negative regulation of replication from the ColE1 origin of replication are employed. Using this process, a yield of 2.2 g of plasmid DNA from a 10 liter-fed batch fermentation were reported. However, the bacterial transformants were grown in the presence of the antibiotic kanamycin which would render them unsuitable for registration and subsequent use in humans. Other approaches have tried to avoid the use of antibiotics; see, e.g., Chen et al., J. Industrial Microbiology and Biotechnology 18 (1997), 43-48. In this report, an automated fed-batch fermentation with feed-back controls based on dissolved oxygen and pH for the production of supercoiled plasmid DNA is disclosed. This DNA is suggested to be useful for DNA vaccines. However, the results reported, for example in FIG. 4, do not support the suggested suitability of the plasmid DNA for vaccination purposes. This is due to the fact that besides ccc plasmid DNA a variety of other plasmid forms are produced under these conditions. Furthermore, this method leads to high contaminations with genomic DNA and, comparatively, only small plasmid amounts can be obtained.
Accordingly, ccc plasmid DNA produced by the prior art methods is unsuitable for a variety of purposes such as medical purposes due to the heterogeneity of the product obtained and/or due to the employment of antibiotics in the production process. The technical problem underlying the present invention was therefore to provide a method that overcomes these prior art difficulties and allows for the production of ccc DNA without the concomitant production of other plasmid forms and which is, moreover, suitable for medical purposes. The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
Thus, the present invention relates to a method for the production of biomass for the isolation of ccc plasmid DNA comprising
(a) culturing a bacterial transformant in a bioreactor containing an antibiotic-free batch-medium comprising
(aa) a carbon source;
(ab) an inorganic salt mixture;
(ac) a nitrogen source;
under batch-culturing conditions;
(b) feeding under feed-back conditions to said culture of (a) at the end of the batch phase, after rising of DO above a threshold-set point, a portion of a feed-medium comprising
(ba) a carbon source; and
(bb) a magnesium salt; and
(c) allowing the bacterial transformant to metabolize said feed-medium.
The term xe2x80x9cbiomassxe2x80x9d, as used in the context of the present invention, relates to any biological material that is or arises from cells or organisms that are capable of reproduction.
The term xe2x80x9cccc plasmid DNAxe2x80x9d refers to a plasmid isoform that is a circular plasmid which is typically but not necessarily underwound relative to a relaxed molecule. This results in a more compact conformity of the molecule which is described as a supercoiled covalently closed circle of the plasmid DNA. In E.coli cells, two enzymes regulate the supercoiling of DNA. The gyrase introduces negative superhelical turns into the molecule while the topoisomerase I relaxes the DNA by introducing single-strand breaks. It is most preferred in accordance with the method of the invention that ccc plasmid DNA in monomeric form is produced. Indeed, the method of the invention provides this particularly desired type of plasmid usually in an amount of more than 90% of overall plasmid production. Also useful, although less preferred, is the production of dimeric ccc plasmid DNA.
The term xe2x80x9cbatch-mediumxe2x80x9d refers to the medium used in batch cultivation, i.e., in discontinuous cultivation of bacterial transformants or other microorganisms. This discontinuous cultivation is characterized by a single inoculation into fresh medium (batch medium) at the start of the cultivation until nutrients and substrates have been exhausted.
The term xe2x80x9cfeed-back conditionsxe2x80x9d relates to the supplementation of medium concentrate during fed-batch cultivation depending on cultivation parameter(s) like, e.g., DO, pH, etc., which are correlated with the growth of the microorganism.
The term xe2x80x9cthreshold-set pointxe2x80x9d in the context of the present invention is intended to mean a defined value for a parameter that is monitored during fed-batch cultivation. In case of over-reaching or under-reaching of that value a monitor signal initiates the response in the regulation of the cultivation. The person skilled in the art is in the position to determine such defined values on the basis of his common knowledge and the teachings of this invention, see, for example, example 1.
The term xe2x80x9cDOxe2x80x9d (concentration of dissolved oxygen) refers hereby to the amount of oxygen in a liquid in per cent of the saturation concentration.
Under the above-defined feed-batch conditions, the concentration of dissolved oxygen (DO) rises during cultivation of bacterial transformants after the consumption of nutrients such as contained in the feed-medium. Therefore, the invention relates in a preferred embodiment to a method wherein the feeding of a bacterial transformant culture comprises repeated feeding-cycles after each rising of the DO above a threshold-set point. It is well known to the person skilled in the art that feeding can be controlled and/or measured via other control parameters, like medium pH, specific growth rate of bacterial transformants, respiration coefficients or others. Nevertheless, all these parameters are interrelated and indicative of the DO. Therefore, irrespective of which parameter is actually employed as a read-out system, it allows a direct (or indirect) conclusion on the DO value. Accordingly, measurement of any of said parameters is covered by the invention as long as it allows a conclusion with respect to the rising of DO above a threshold-set point.
The term xe2x80x9callowing the bacterial transformant to metabolize said feed-mediumxe2x80x9d relates to the partial or complete metabolization of said feed-medium and preferably to the essentially complete metabolization of said feed medium.
In accordance with the present invention it has been found that the here disclosed method leads to the production of high plasmid concentrations and to plasmids with a DNA homogeneity of typically more than 90% ccc monomers. The result is all the more surprising since ccc monomers of the indicated high homogeneity are obtained in the absence of selection pressure by antibiotics. The person skilled in the art is able to employ the here disclosed method for the production of ccc DNA over a broad range of types or sizes of plasmids. The method of the invention in addition allows the isolation of a larger quantity of the desired plasmid from bacterial cultures than was possible with prior art methods. This also leads to a significant cost reduction of the fermentation processes since smaller fermentors may be employed as compared to prior art processes if the same amount of plasmid is to be generated.
A further significant advantage of the method of the present invention for the production of biomass for the isolation of ccc plasmid DNA relates to the fact that the media are devoid of antibiotics. The obtained ccc DNA is thus definitively antibiotic-free and may, without time consuming and costly further elaborate purification schedules, be employed in medical therapy where antibiotic contaminations are to be avoided.
It is most preferred in the method of the present invention to employ batch-or feed media devoid of yeast extract or other complex amino acid sources derived from natural sources. This is because the cultivation in such fully synthetic media does not bear the danger of source related contamination and has the advantage of a higher reproducibility. Another preferred option would be to use semi-synthetic media, comprising, for example, yeast extracts, plant extracts, peptone supplements and others. These synthetic or semi-synthetic media may be employed in one or more steps of the culturing of the transformants. This also includes the pre-culturing step addressed below. It is also envisaged by the present invention to cultivate in/feed different types of media at different cultivation steps. Different media may also be fed when repeated feeding cycles are employed as discussed herein below. It is most preferred that these media are autoclavable. Magnesium salts should be autoclaved separately.
As has been outlined above, in a preferred embodiment of the method of the invention, step (b) comprises repeated feeding cycles after each rising of the DO above a threshold-set point. This embodiment of the invention is particularly advantageous since it allows the production of high yields of biomass which allows for the isolation of high amounts of ccc-plasmid DNA.
The bacterial transformant used in the method of the invention can be either a gram-negative or a gram-positive bacterium. Examples of gram-positive bacteria are bacteria of the genus Bacillus, for example Bacillus subtilis. In a preferred embodiment, the bacterial transformant is an E. coli cell.
Whereas the person skilled in the art is capable of devising or preparing a suitable carbon source, glycerol is preferably used as a carbon source in the method of the present invention.
A variety of well known and established nitrogen sources may be employed in the method of the invention. In a further preferred embodiment of the present invention, the nitrogen source employed is NH3.
In a further preferred embodiment of the method of the invention the carbon source in the batch-medium is in a (initial final) concentration of xe2x89xa6100 g/l.
In another preferred embodiment of the method of the invention the carbon source in the feed-medium is in a (initial final) concentration of xe2x89xa61000 g/l.
In yet another preferred embodiment of the method of the invention the nitrogen source is in a (initial final) concentration of xe2x89xa630%.
In a further preferred embodiment of the method of the invention the inorganic salt mixture comprises Na2HPO4xe2x89xa66 g/l, KH2PO4xe2x89xa63 g/l, NaClxe2x89xa60,5 g/l, and citric acid.H2Oxe2x89xa61,5 g/l. This refers to the initial final concentration in the medium, i.e. the final concentration that is present in the medium at the onset of fermentation.
Most preferably, said inorganic salt mixture also comprises a magnesium salt, preferably MgSO4, for example complexed with water. In this case, an initial concentration of smaller than or about 0.3 g/l is preferred. It is also desired that the magnesium salt is autoclaved separately.
In another preferred embodiment of the method of the invention in step (b) the magnesium salt concentration is in a range of 5-100 mM. This again refers to the initial final concentration in the medium.
In a particularly preferred embodiment of the method of the invention in step (b) the magnesium salt concentration is 80 mM. In accordance with the invention, it has surprisingly been found that in particular rather high magnesium salt concentrations such as 80 mM, preferrebly of MgSO4, yield excellent results with regard to the homogeneity of the ccc plasmid monomers.
Magnesium salts that may be used in accordance with the present invention comprises MgCl2, Mg(NO3)2, MgSO4 or others. In a preferred embodiment of the method of the invention the magnesium salt is MgSO4.
In another preferred embodiment of the invention a solution of trace elements is added in steps (a) and/or (b). Addition of trace elements may further enhance the plasmid yield.
In another embodiment of the method of the invention the solution of trace elements comprises each of the following compounds
FeCl3.6H2O in a final concentration of preferably xe2x89xa654.0 mg/l
ZnSO4.7H2O in a final concentration of preferably xe2x89xa613.8 mg/l
MnSO4.H2O in a final concentration of preferably xe2x89xa618.5 mg/l
CoSO4.7H2O in a final concentration of preferably xe2x89xa65.6 mg/l
CuCl2 in a final concentration of preferably xe2x89xa61.7 m/l
H3BO3 in a final concentration of preferably xe2x89xa610 mg/l
Na2MoO4.2H2O in a final concentration of preferably xe2x89xa625 mg/l; and
citric acid in a final concentration of preferably xe2x89xa650 mg/l
Bacterial or prokaryotic growth media can advantageously be supplemented with an amino acid source. Therefore, in a further preferred embodiment of the method of the invention, the batch-medium comprises an amino acid source.
In another preferred embodiment of the method of the invention the feed-medium comprises an amino acid source. Amino acid sources are well known to the person skilled in the art and can comprise yeast extracts, plant extracts, peptone supplements and others (see Sambrook et al., loc. cit.).
In a further embodiment of the method of the invention the culturing of the bacterial transtormant is carried out at a temperature range of 30xc2x0 C. to 42xc2x0 C.
In a particularly preferred embodiment of the method of the invention the temperature range is about 35xc2x0 C. to 38xc2x0 C.
In order to compensate auxotrophic requirements of bacterial transformants, it is preferred that in the method of the invention the batch-medium comprises a bacterial host strain specific supplement. A variety of specific supplements for different bacterial host strains has been described and is well known in the art, e.g., thiamine for bacterial transformants carrying a thiamine deficiency.
In a further preferred embodiment of the method of the invention the host cells are harvested, after step (c), from said cultures. Harvesting of bacterial transformants is one of the conventional methods in fermentation and molecular biology techniques. The harvest of transformants can comprise filtering, centrifugation or similar methods which are well known in the art.
In yet another preferred embodiment of the method of the invention, the host cells are, after step (c), subjected to a washing step before or after harvesting. These washing steps can be carried out in a solution that does not affect the integrity of the cells but removes culturing compounds from the cell.
In another preferred embodiment of the method of the invention, a further step comprises the freezing or freeze-drying of the transformants after step (c) or after the steps identified herein above in any of the further preferred embodiments. The embodiment is particularly useful if an immediate isolation of ccc plasmid is not desired. Frozen or freeze-dried cells may be conveniently be shipped or stored until further use.
In yet another preferred embodiment of the method of the invention a further step comprises the isolation of ccc DNA.
There are multiple ways to isolate ccc DNA which are well know to the person skilled in the art. These include CsCl gradient centrifugation and chromatography purification methods (see, e.g., Sambrook et al., xe2x80x9cMolecular Cloning, A Laboratory Manualxe2x80x9d, CSH Press, 2nd edition, 1989, Cold Spring Harbor N.Y.).
As stated herein above, isolated plasmid DNA consisting of more than 90% ccc monomers may be obtained. The person skilled in the art, when employing the teachings of the present invention, is able to obtain ccc DNA in large quantities which fulfills the quality criteria of plasmid DNA for gene therapeutic and nucleic acid vaccination approaches (see Schorr et al., DNA Vaccines 772 (1995), 271-273) without subsequent exhaustive purification steps to get rid, for example, of antibiotics. Thus, it is possible to employ the obtained ccc DNA of the invention for nucleic acid vaccinations as described in the prior art, for example in Davis et al., Vaccine 12 (1994), 1503-1509, or for gene therapeutic strategies as disclosed in Cao et al., Human Gene Therapy 6 (1995), 1497-1501.
Furthermore, in another preferred embodiment, the invention relates to a method further comprising the step of (axe2x80x2) pre-culturing the bacterial transformant in an antibiotic-free medium.
In a particularly preferred embodiment of the method of the invention the bacterial transformant is in exponential growth phase after the end of said pre-culturing. The exponential growth phase may be assessed by conventional technology, for example, by determining the optical density of the culture broth.